Murine Model of Candida albicans and Streptococcus agalactiae Vaginal Co-Colonization

Summary

This protocol details a method to vaginally co-colonize mice with Candida albicans and Streptococcus agalactiae. It may be used to study polymicrobial interactions, host-microbe interactions, and host responses to these organisms.

Abstract

Diverse microbes are isolated from the human vaginal tract, and many of the organisms that inhabit this niche can switch between commensal and pathogenic lifestyles. Factors, including community composition and the host environment, influence disease outcomes; however, the mechanisms determining these outcomes are not well understood. In this manuscript, we describe a murine model to investigate interkingdom interactions and the host response to Candida albicans and Streptococcus agalactiae (Group B Streptococcus, GBS), pathobionts that colonize the female genital tract (FGT). Emerging evidence suggests that C. albicans colonization is a risk factor for GBS carriage due to their frequent co-isolation. During pregnancy, GBS colonization can lead to adverse outcomes including chorioamnionitis and stillbirth. GBS can also be transmitted to a fetus in utero or a neonate during birth and is a leading cause of neonatal meningitis. Colonization of the vaginal tract is a critical precursor to GBS disease, and this niche exerts numerous selective pressures, some of which may be influenced by C. albicans colonization. This protocol describes a model of concurrent C. albicans-GBS vaginal co-colonization using antibiotic treatment and a single dose of 17β-estradiol to establish C. albicans colonization, followed by the removal of antibiotics and inoculation with GBS. Microbial burdens in the vaginal lumen are quantified over the course of the experiment by lavage, and burdens throughout the FGT are quantified by dissection, homogenization, and plating of the vagina, cervix, and uterus. This method will enable investigation of the mechanisms by which direct and indirect polymicrobial interactions influence the lifestyles and pathogenesis of these two organisms, as well as the host immune landscape in the FGT.

Introduction

Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterial pathobiont that colonizes multiple polymicrobial sites, including the gastrointestinal and vaginal tracts. While vaginal colonization is typically asymptomatic, during pregnancy, GBS can ascend from the vaginal tract into the uterus and traverse intact membranes, causing infection of the amniotic fluid and placenta. Thus, it is a primary risk factor for adverse pregnancy outcomes, fetal infection, and neonatal disease^1,2. As a leading cause of invasive infection in neonates, it is critical to characterize facto....

Protocol

The animal work presented in this manuscript was approved by and performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Anschutz under protocol #00316. In the timeline below, day 0 refers to the day of S. agalactiae inoculation.

1. Administer antibiotics before inoculation.

1. Make a concentrated solution of 50 mg/mL each of ampicillin, neomycin, and gentamycin dissolved in mouse drinking water. Use a 0.22 µm filter to sterilize the antibiotic solution.
2. Add the antibiotic solution to the mouse drinking water at a 1:100 dilution by a....

Results

To characterize GBS and C. albicans vaginal colonization, mice were intravaginally inoculated with C. albicans strain SC5314 or a PBS vehicle control, followed by inoculation with GBS strain COH1. This strain represents hypervirulent capsular serotype III, sequence type 17 isolates that are most frequently associated with neonatal invasive disease and meningitis¹. Mice not colonized with GBS one day post inoculation were excluded from analysis. Over a four-day experimental timeli.......

Discussion

GBS vaginal colonization poses a critical risk to maternal-fetal and neonatal health. In vivo models are imperative for understanding the determinants of colonization and for developing preventative therapeutics. In this manuscript, we describe a murine model to vaginally co-colonize mice with GBS and C. albicans, assess microbial burdens in the vaginal lumen and FGT, and collect relevant tissues for further downstream analyses. This model has been developed to study fungal-bacterial and host-microbe in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 µm filter	Sigma-Aldrich	SLGSR33SB
0.45 µm filter	Sigma-Aldrich	SLHAR33SB
1 mL luer lock syringe	McKesson	16-S1C
1.0 mm zirconia beads	Research Products International	9835
10 mL luer lock syringe	McKesson	16-S10C
17β-estradiol	Sigma-Aldrich	E8875	CAUTION: 17β-estradiol can be absorbed through skin and mucosal surfaces. Wear appropriate personal protective equipment (PPE) and exercise caution while using.
18G 1.5" needle	BD	305196
2 mL screw-cap tube	Fisher Scientific	02-681-374
26G 0.5" needle	McKesson	16-N2605
Agar	Alpha Biosciences	A01-102
Ampicillin	Research Products International	A40040
CHROMagar Candida	CHROMagar	CA222
CHROMagar StrepB	CHROMagar	SB282
Gentamycin	VWR Life Science	0304
Neomycin	Research Products International	N20040
Phosphate-buffered saline	Fisher Scientific	BP2944
Sesame oil	Sigma-Aldrich	S3547
Todd Hewitt Broth	Research Products International	T47500
Yeast Peptone Dextrose Broth	BD	242810